Methods and Compositions for Correlating Genetic Markers with Prostate Cancer Risk

ABSTRACT

The present invention provides methods of assessing an individual subject&#39;s risk of developing prostate cancer, comprising: a) analyzing a nucleic acid sample obtained from the subject and determining a genotype for the subject at a plurality of biallelic polymorphic loci, wherein each of said plurality has an associated allele and an unassociated allele, wherein the genotype is selected from the group consisting of homozygous for the associated allele, heterozygous, and homozygous for the unassociated allele; and b) calculating a cumulative relative risk (CRR) for the subject based on the genotype determined in step (a). A CRR of greater than 1.00 identifies a subject as having an increased risk of developing prostate cancer and also can identify a subject who is a candidate for early PSA screening, prostate biopsy and/or chemoprevention.

STATEMENT OF PRIORITY

This application claims the benefit, under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 61/379,965, filed Sep. 3, 2010, theentire contents of which are incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

Aspects of the present invention were made with government support underGrant No CA148463 from the National Cancer Institute. The United StatesGovernment has certain rights to this invention.

FIELD OF THE INVENTION

The present invention provides methods and compositions directed toassessing risk of having or developing prostate cancer by analyzingmultiple single nucleotide polymorphisms in nucleic acid of a subject.

BACKGROUND OF THE INVENTION

Prostate cancer (PCa) is the most common solid organ malignancyaffecting American men and the second leading cause of cancer relateddeath. Approximately one million prostate biopsies are performed yearlyin the U.S. The vast majority of these biopsies are performed due toelevated levels of the PCa marker prostate-specific antigen (PSA).However, only a quarter of these biopsies result in a diagnosis of PCa,highlighting the inadequate performance of currently availableparameters such as PSA to predict PCa. Persistently elevated PSA levelsand/or other clinical parameters that prompted initial biopsiescontribute to stress and anxiety among both patients and theirurologists. Thus, the predictive performance of currently availableclinical parameters such as PSA is limited. Furthermore, management ofmen following negative prostate biopsy for prostate cancer ischallenging. Novel biomarkers are urgently needed to better determinethe need for initial and repeat prostate biopsy and assess anindividual's risk.

Single nucleotide polymorphisms (SNPs) are stable genetic markersthroughout the human genome, which can be tested for their associationwith various disease traits. These markers can be tested at birth andwill not change in a patient's lifetime and thus represent a new form ofbiomarkers that predict lifetime risk to disease as opposed to animmediate risk.

Numerous PCa risk-associated single nucleotide polymorphisms (SNPs) havebeen discovered from genome-wide association studies (GWAS). To date, 33SNPs have been consistently found, in several populations of Caucasianrace, to be associated with prostate cancer (PCa) risk (Table 1). Theserisk-associated SNPs have been consistently replicated in multiplecase-control study populations of European descent. Although each ofthese SNPs is only moderately associated with PCa risk, a genetic scorebased on a combination of risk-associated SNPs can be used to identifyan individual's risk for PCa. These risk-associated SNPs have broadpractical applications because they are common in the generalpopulation.

The present invention overcomes previous shortcomings in the art byidentifying significant statistical associations between multiplegenetic markers and prostate cancer risk.

SUMMARY OF THE INVENTION

The present invention provides a method of identifying a subject ashaving an increased risk of developing prostate cancer, comprising: a)determining, from a nucleic acid sample obtained from the subject, agenotype for the subject at a plurality of biallelic polymorphic loci,wherein each of said plurality has an associated allele and anunassociated allele, wherein the genotype is selected from the groupconsisting of homozygous for the associated allele, heterozygous, andhomozygous for the unassociated allele; and b) calculating a cumulativerelative risk (CRR, also known as genetic score) for the subject basedon the genotype determined in step (a), wherein a cumulative relativerisk of greater than 1.0 identifies the subject as having an increasedrisk of developing prostate cancer. The step of determining includesmanipulating a fluid or tissue sample obtained from the subject toextract nucleic acid of the subject from the sample in a form thatallows for the nucleotide sequence of the nucleic acid to be identified.

In the methods of this invention, identification of the subject'sincreased risk of developing prostate cancer can also includesinformation about the subject's family history, prostate specificantigen (PSA) level, free to total PSA ratio, age, prostate volume,prior prostate biopsy history, number of previous biopsy cores and/orfamily history. Such information can, for example, be identified inquantitative terms that can be incorporated into the calculationsdescribed herein to determine how these factors influence the subject'srisk of developing prostate cancer. Thus, in some embodiments, thesubject can have a family history of prostate cancer or the subject mayhave no family history of prostate cancer. In some embodiments, thesubject may have never had a prostate biopsy and in some embodiments,the subject may have had a prior negative prostate biopsy. In furtherembodiments, the subject may have had a prior positive prostate biopsy.

The methods of this invention have utility in guiding the subject andhis clinician in determining courses of action for treating orpreventing or monitoring the occurrence of prostate cancer. Thus, insome embodiments, the identification of the subject as having anincreased risk of developing prostate cancer identifies the subject as acandidate for prostate serum antigen (PSA) screening prior to age 50.Thus, due to the subject's increased risk of developing prostate cancer,such screening at an early age may allow for the detection of prostatecancer at is onset or at an early stage when it can be readily treated.

In further embodiments, identification of the subject as having anincreased risk of developing prostate cancer according to the methods ofthis invention identifies the subject as a candidate for prostatebiopsy. In particular embodiments, a subject with a CRR of greater than1.00, together with other clinical variables, such as PSA, prostatevolume and digital rectal exam (DRE) is a subject who is a goodcandidate for prostate biopsy. Thus, due to the subject's increased riskof developing prostate cancer, such a biopsy may allow for the detectionof prostate cancer at is onset or at an early stage when it can bereadily treated.

In yet further embodiments, identification of the subject as having anincreased risk of developing prostate cancer according to the methods ofthis invention identifies a subject who has had a prior negativeprostate biopsy as a candidate for a subsequent or repeat biopsyprostate biopsy. Thus, due to the subject's increased risk of developingprostate cancer, such a biopsy may allow for the detection of prostatecancer at is onset or at an early stage when it can be readily treated.

In additional embodiments, identification of the subject as having anincreased risk of developing prostate cancer according to the presentinvention identifies the subject as a candidate for chemopreventivetherapy, such as, for example, a 5-alpha reductase inhibitor (e.g.,dutasteride; finasteride) as is known in the art. In particularembodiments, a subject with a CRR of greater than 1.00 and/or a positivefamily history of prostate cancer should be considered forchemoprevention.

In the methods of this invention, the plurality of biallelic polymorphicloci employed in the methods of this invention is a multiplicity (e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33), in any combination,of the 33 single nucleotide polymorphisms of Table 1. In someembodiments, the plurality or biallelic polymorphic loci employed in themethods of this invention is the 33 single nucleotide polymorphisms ofTable 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Detection rates for prostate cancer for men below or above themedian estimated risk based on a) the genetic model (genetic score of 33PCa risk-associated SNPs) and b) the best clinical model (with fiveparameters: age, family history, free/total PSA ratio, prostate volume,and number of cores at initial biopsy). Detection rates for the geneticmodel were directly estimated. Detection rates for the best clinicalmodel were estimated based on four-fold cross validation. Vertical linesin each bar represent 95% CI of detection rates.

FIG. 2. Detection rates for prostate cancer for men below or above themedian estimated risk based on the best clinical model (age, familyhistory, free/total PSA ratio, prostate volume, and number of cores atinitial biopsy), and stratified by genetic risk (lower or higher half ofgenetic risk). Vertical lines in each bar represent 95% CI of detectionrates.

FIG. 3. Detection rates for high-grade prostate cancer for men below orabove the median estimated risk based on a) the genetic model, b) thebest clinical model (age, family history, free/total PSA ratio, prostatevolume, and number of cores at initial biopsy), and c) the best clinicalmodel and stratified by genetic risk (lower or higher half of geneticrisk). Vertical lines in each bar represent 95% CI of detection rates.

FIGS. 4 a-f. Detection rate of PCa and high grade PCa among men withvarious estimated PCa risk based on genetic score, clinical variablesand combination of both.

FIGS. 5 a-b. Detection rate of PCa and high-grade PCa among men withvarious estimated PCa risk based on the best clinical variables,stratified by genetic risk.

FIGS. 6 a-f. FIGS. 6 a, b and c show the distribution of estimated riskfor each of the three models. These models consist of genetic score(GS), GS plus three pre-biopsy variables (GS+3 variables), and GS plusthree pre-biopsy and 3 post-biopsy variables (GS+5 variables). FIGS. 6d, e and f show that, for each respective model (GS, GS+3, GS+5), thePCa detection rate trends upward in reflection of increasing riskquartile.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure, which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

The present invention is based on the unexpected discovery of a methodof predicting PCa risk in an individual, based on an assessment of theindividual's genotype at a multiplicity (e.g., any 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32 or 33, in any combination) of the 33 SNPs ofTable 1. In some embodiments, the method can include an assessment of anindividual's genotype at all 33 SNPs of Table 1. In some embodiments,the method can also include an assessment of an individual's genotype atany SNP site in linkage disequilibrium (LD) with any of the 33 SNPs inTable 1. This method, which is called PCS33, provides a powerfulpredictor of PCa risk. This predictor out-performs any of the currentlyavailable parameters of PCa risk as assessed in a unique studypopulation (Table 2). In addition, this predictor can improve theability of a collection of currently available parameters to predict anyPCa risk. Furthermore, this test can be used alone, to identify higherrisk individuals who wish to pursue PCa screening or together withestablished predictors to identify men who may warrant an initial orrepeat prostate biopsy. The output of the test can be a cumulativerelative risk (CRR, an estimated risk based on the individual's genotypeat a multiplicity, in any combination, of these 33 SNPs, which is arelative risk based on genotype with respect to the general population),a percentile risk (risk level in percentile in the distribution of thepopulation risk to PCa), absolute risk (risk of PCa over time), or PCarisk score (probability of being diagnosed with PCa as determined by alogistic regression model). There is no true normal value for this test,which allows for the patient or treating physician to determine the risklevel which is clinically meaningful to that particular individual. Riskin the general population can be determined, for example, from suchsources as surveillance, epidemiology and end results (SEER)information, available on the internet at http://seer.cancer.gov.

Thus, in one aspect, the present invention provides a method ofassessing a subject's risk of having or developing prostate cancer bycarrying out an assessment of the subject's genotype at all of the 33SNP sites or a multiplicity, in any combination, of the 33 SNP siteslisted in Table 1 (e.g., a PCS33 risk assessment) according to themethods described herein.

In some embodiments, the PCS33 risk assessment can be used by itself topredict a subject's risk for PCa, which may direct the subject's desireto pursue PCa screening or alter the frequency of PCa screening.

In further embodiments, the PCS33 risk assessment can be used incombination with known clinical variables (prostate specific antigen(PSA), free to total PSA ratio, age, and/or family history) to predict asubject's risk for PCa. This may help urologists and their patientsdecide whether to pursue prostate biopsy in men who have never had aprior prostate biopsy.

In yet further embodiments, the PCS33 risk assessment can be used incombination with known clinical variables following negative prostatebiopsy (prostate volume, number of previous biopsy cores, PSA, free tototal PSA ratio, age, and/or family history) to predict a subject's riskfor PCa. This may help urologists and their patients decide whether topursue repeat prostate biopsy in men who have had a prior negativeprostate biopsy.

The risk assessment provided to the patient subjects and their treatingurologist may include any or all of the following.

1. Cumulative relative risk (CRR) to PCa. The CRR to PCa provided to thesubject is derived by obtaining the subject's genotype at the 33 SNPs ofTable 1 and may in addition include information on clinical parametersshould they be available. For the genetic component of the CRR (CRR),allelic odds ratios (ORs) are obtained from meta-analyses which are thenused to determine a relative risk to the general population for aparticular genotype at a particular SNP for an individual. The CRR basedon 33 SNPs or a multiplicity, in any combination, of the 33 SNPs is thengenerated by multiplying the relative risks for each of the SNPs for agiven individual. This is the genetic component of the CRR to PCapresented to the subject and represents the fold increase in PCa riskcompared to the general population. A similar analysis may be performedincluding the ORs and relative risks for each available clinicalparameter based on the outlined study population and then can be usedwith the genetic component to provide an overall CRR to PCa.

2. Percentile risk to PCa. The percentile risk is generated bydetermining the risk level in percentile in the distribution ofpopulation relative risk for PCa.

3. Absolute risk to PCa. Absolute risk is determined by taking intoconsideration the CRR and incidence and mortality rates from PCa andmortality due to other causes. This describes the PCa risk over time andfor the purposes of this invention, represents the lifetime risk of PCa.

4. PCa risk score. PCa risk score is another means to measure theprobability of being diagnosed with PCa. It does not take intoconsideration time or population parameters such as disease incidence ormortality rates. It is generated by fitting the CRR from the geneticcomponent alone or in combination with other predictors (includinggenetic score, PSA, F/T PSA ratio, family history of PCa, age), into alogistic regression model.

DEFINITIONS

As used herein, “a,” “an” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

As used herein, the term “prostate cancer” or “PCa” describes anuncontrolled (malignant) growth of cells originating from the prostategland, which is located at the base of the urinary bladder and isresponsible for helping control urination as well as forming part of thesemen. Symptoms of prostate cancer can include, but are not limited to,urinary problems (e.g., not being able to urinate; having a hard timestarting or stopping the urine flow; needing to urinate often,especially at night; weak flow of urine; urine flow that starts andstops; pain or burning during urination), difficulty having an erection,blood in the urine and/or semen, and/or frequent pain in the lower back,hips, and/or upper thighs.

As used herein, the term “aggressive prostate cancer” means prostatecancer that is poorly differentiated, having a Gleason grade of 7 orabove and an “indolent prostate cancer” having a Gleason grade of 6. TheGleason grading system is the most commonly used method for grading PCa.

All the SNP positions described herein are based on Build 36.

Also as used herein, “linked” describes a region of a chromosome that isshared more frequently in family members or members of a populationmanifesting a particular phenotype and/or affected by a particulardisease or disorder, than would be expected or observed by chance,thereby indicating that the gene or genes or other identified marker(s)within the linked chromosome region contain or are associated with anallele that is correlated with the phenotype and/or presence of adisease or disorder (e.g., aggressive PCa), or with an increased ordecreased likelihood of the phenotype and/or of the disease or disorder.Once linkage is established, association studies can be used to narrowthe region of interest or to identify the marker (e.g., allele orhaplotype) correlated with the phenotype and/or disease or disorder.

Furthermore, as used herein, the term “linkage disequilibrium” or “LD”refers to the occurrence in a population of two or more (e.g., 3, 4, 5,6, 7, 8, 9, 10, etc.) linked alleles at a frequency higher or lower thanexpected on the basis of the gene frequencies of the individual genes.Thus, linkage disequilibrium describes a situation where alleles occurtogether more often than can be accounted for by chance, which indicatesthat the two or more alleles are physically close on a DNA strand.

The term “genetic marker” or “polymorphism” as used herein refers to acharacteristic of a nucleotide sequence (e.g., in a chromosome) that isidentifiable due to its variability among different subjects (i.e., thegenetic marker or polymorphism can be a single nucleotide polymorphism,a restriction fragment length polymorphism, a microsatellite, a deletionof nucleotides, an addition of nucleotides, a substitution ofnucleotides, a repeat or duplication of nucleotides, a translocation ofnucleotides, and/or an aberrant or alternate splice site resulting inproduction of a truncated or extended form of a protein, etc., as wouldbe well known to one of ordinary skill in the art).

A “single nucleotide polymorphism” (SNP) in a nucleotide sequence is agenetic marker that is polymorphic for two (or in some case three orfour) alleles. SNPs can be present within a coding sequence of a gene,within noncoding regions of a gene and/or in an intergenic (e.g.,intron) region of a gene. A SNP in a coding region in which both formslead to the same polypeptide sequence is termed synonymous (i.e., asilent mutation) and if a different polypeptide sequence is produced,the alleles of that SNP are non-synonymous. SNPs that are not in proteincoding regions can still have effects on gene splicing, transcriptionfactor binding and/or the sequence of non-coding RNA.

The SNP nomenclature provided herein refers to the official ReferenceSNP (rs) identification number as assigned to each unique SNP by theNational Center for Biotechnological Information (NCBI), which isavailable in the GenBank® database.

In some embodiments, the term genetic marker is also intended todescribe a phenotypic effect of an allele or haplotype, including forexample, an increased or decreased amount of a messenger RNA, anincreased or decreased amount of protein, an increase or decrease in thecopy number of a gene, production of a defective protein, tissue ororgan, etc., as would be well known to one of ordinary skill in the art.

An “allele” as used herein refers to one of two or more alternativeforms of a nucleotide sequence at a given position (locus) on achromosome. An allele can be a nucleotide present in a nucleotidesequence that makes up the coding sequence of a gene and/or an allelecan be a nucleotide in a non-coding region of a gene (e.g., in a genomicsequence). A subject's genotype for a given gene is the set of allelesthe subject happens to possess. As noted herein, an individual can beheterozygous or homozygous for any allele of this invention.

Also as used herein, a “haplotype” is a set of alleles on a singlechromatid that are statistically associated. It is thought that theseassociations, and the identification of a few alleles of a haplotypeblock, can unambiguously identify all other alleles in its region. Theterm “haplotype” is also commonly used to describe the geneticconstitution of individuals with respect to one member of a pair ofallelic genes; sets of single alleles or closely linked genes that tendto be inherited together.

The terms “increased risk” and “decreased risk” as used herein definethe level of risk that a subject has of developing prostate cancer, ascompared to a control subject that does not have the polymorphisms andalleles of this invention in the control subject's nucleic acid.

A sample of this invention can be any sample containing nucleic acid ofa subject, as would be well known to one of ordinary skill in the art.Nonlimiting examples of a sample of this invention include a cell, abody fluid, a tissue, biopsy material, a washing, a swabbing, etc., aswould be well known in the art.

A subject of this invention is any animal that is susceptible toprostate cancer as defined herein and can include, for example, humans,as well as animal models of prostate cancer (e.g., rats, mice, dogs,nonhuman primates, etc.). In some aspects of this invention, the subjectcan be Caucasian (e.g., white; European-American; Hispanic), as well asof black African ancestry (e.g., black; African American;African-European; African-Caribbean, etc.) or Asian. In further aspectsof this invention, the subject can have a family history of prostatecancer or aggressive prostate cancer (e.g., having at least one firstdegree relative having or diagnosed with prostate cancer or aggressiveprostate cancer) and in some embodiments, the subject does not have afamily history of prostate cancer or aggressive prostate cancer.Additionally a subject of this invention can have a diagnosis ofprostate cancer in certain embodiments and in other embodiments, asubject of this invention does not have a diagnosis of prostate cancer.In yet further embodiments, the subject of this invention can have anelevated prostate-specific antigen (PSA) level and in other embodiments,the subject of this invention can have a normal or non-elevated PSAlevel. In some embodiments, the PSA level of the subject may not beknown and/or has not been measured.

As used herein, “nucleic acid” encompasses both RNA and DNA, includingcDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNAand chimeras, fusions and/or hybrids of RNA and DNA. The nucleic acidcan be double-stranded or single-stranded. Where single-stranded, thenucleic acid can be a sense strand or an antisense strand. In someembodiments, the nucleic acid can be synthesized using oligonucleotideanalogs or derivatives (e.g., inosine or phosphorothioate nucleotides,etc.). Such oligonucleotides can be used, for example, to preparenucleic acids that have altered base-pairing abilities or increasedresistance to nucleases.

An “isolated nucleic acid” is a nucleotide sequence that is notimmediately contiguous with nucleotide sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived orin which it is detected or identified. Thus, in one embodiment, anisolated nucleic acid includes some or all of the 5′ non-coding (e.g.,promoter) sequences that are immediately contiguous to a codingsequence. The term therefore includes, for example, a recombinant DNAthat is incorporated into a vector, into an autonomously replicatingplasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (e.g., a cDNA or a genomic DNAfragment produced by PCR or restriction endonuclease treatment),independent of other sequences. It also includes a recombinant DNA thatis part of a hybrid nucleic acid encoding an additional polypeptide orpeptide sequence.

The term “isolated” can refer to a nucleic acid or polypeptide that issubstantially free of cellular material, viral material, and/or culturemedium (e.g., when produced by recombinant DNA techniques), or chemicalprecursors or other chemicals (when chemically synthesized). Moreover,an “isolated fragment” is a fragment of a nucleic acid or polypeptidethat is not naturally occurring as a fragment and would not be found inthe natural state.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout five nucleotides to about 500 nucleotides (e.g. 5, 6, 7, 8, 9, 10,12, 15, 18, 20, 21, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500nucleotides). In some embodiments, for example, an oligonucleotide canbe from about 15 nucleotides to about 30 nucleotides, or about 20nucleotides to about 25 nucleotides, which can be used, for example, asa primer in a polymerase chain reaction (PCR) amplification assay and/oras a probe in a hybridization assay or in a microarray. Oligonucleotidesof this invention can be natural or synthetic, e.g., DNA, RNA, PNA, LNA,modified backbones, etc., as are well known in the art.

The present invention further provides fragments of the nucleic acids ofthis invention, which can be used, for example, as primers and/orprobes. Such fragments or oligonucleotides can be detectably labeled ormodified, for example, to include and/or incorporate a restrictionenzyme cleavage site when employed as a primer in an amplification(e.g., PCR) assay.

The detection of a polymorphism, genetic marker or allele of thisinvention can be carried out according to various protocols standard inthe art and as described herein for analyzing nucleic acid samples andnucleotide sequences, as well as identifying specific nucleotides in anucleotide sequence.

For example, nucleic acid can be obtained from any suitable sample fromthe subject that will contain nucleic acid and the nucleic acid can thenbe prepared and analyzed according to well-established protocols for thepresence of genetic markers according to the methods of this invention.In some embodiments, analysis of the nucleic acid can be carried byamplification of the region of interest according to amplificationprotocols well known in the art (e.g., polymerase chain reaction, ligasechain reaction, strand displacement amplification, transcription-basedamplification, self-sustained sequence replication (3SR), Qβ replicaseprotocols, nucleic acid sequence-based amplification (NASBA), repairchain reaction (RCR) and boomerang DNA amplification (BDA), etc.). Theamplification product can then be visualized directly in a gel bystaining or the product can be detected by hybridization with adetectable probe. When amplification conditions allow for amplificationof all allelic types of a genetic marker, the types can be distinguishedby a variety of well-known methods, such as hybridization with anallele-specific probe, secondary amplification with allele-specificprimers, by restriction endonuclease digestion, and/or byelectrophoresis. Thus, the present invention further providesoligonucleotides for use as primers and/or probes for detecting and/oridentifying genetic markers according to the methods of this invention.

In some embodiments of this invention, detection of an allele orcombination of alleles of this invention can be carried out by anamplification reaction and single base extension. In particularembodiments, the product of the amplification reaction and single baseextension is spotted on a silicone chip.

In yet additional embodiments, detection of an allele or combination ofalleles of this invention can be carried out by matrix-assisted laserdesorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS).

It is further contemplated that the detection of an allele orcombination of alleles of this invention can be carried out by variousmethods that are well known in the art, including, but not limited tonucleic acid sequencing, hybridization assay, restriction endonucleasedigestion analysis, electrophoresis, and any combination thereof.

The present invention further comprises a kit or kits to carry out themethods of this invention. A kit of this invention can comprisereagents, buffers, and apparatus for mixing, measuring, sorting,labeling, etc, as well as instructions and the like as would beappropriate for genotyping the 33 SNPs of Table 1 in a nucleic acidsample. The kit may further comprise control reagents, e.g., to identifymarkers for a specific ethnicity or gender.

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES Example 1 Patient A

A 40 year old Caucasian man with a significant family history ofprostate cancer with his father and paternal grandfather dying of thedisease, sees his primary care physician, asking him about PCa risk andif and/or when to begin prostate cancer screening. He is referred to hisurologist who counsels him about the risks and benefits of prostatecancer screening and offers him a genetic test based on 33 SNPs whichcan measure his baseline risk for PCa. He accepts and has a sample ofhis nucleic acid tested.

He sees his urologist who goes over the report of the genetic test,which describes the patient's risk for PCa based only on his geneticprofile in several formats:

-   -   CRR: 2.40    -   Percentile risk: 96^(th) percentile    -   Absolute risk: 0.31    -   PCa risk score: 0.37

Given the above report, the patient comes to the conclusion that he isat high risk for PCa and decides to pursue PSA-based PCa screening. Thisis based on the fact that he is at 2.4 fold increase in risk for PCa ascompared to the general population and that only 4% of the populationhas a higher risk for prostate cancer. Furthermore, for him, a lifetime,absolute risk of 31% is high and warrants follow-up. To date, there isno well established clinical parameter applicable to the 40 year oldmale with the exception of possibly family history. We have alreadydemonstrated (Table 2) that the genetic test outperforms family history,the only other potentially applicable existing clinical predictor of PCarisk. In addition, family history may not be available in some casessuch as adoption, lack of male family members and a lack ofcommunication between family members.

Example 2 Patient B

Patient A's 50 year old brother heard from his brother about theabove-described genetic test for PCa risk. He had his first ever PSA,which was borderline high at 4.0 ng/ml. He has heard that prostatebiopsy is very uncomfortable and would like to avoid it if at allpossible. Based on his initial clinical parameters, his urologist offershim a prostate biopsy. He undergoes the genetic testing, which gives himthe following result:

-   -   CRR: 0.90    -   Percentile risk: 49^(th) percentile    -   Absolute risk: 0.12    -   PCa risk score: 0.19

On the basis of this report, he concludes that he is at lower risk forPCa and opts to continue to follow his PSA as opposed to proceedingdirectly to prostate biopsy. He makes this decision based on thesubjective judgment that, for him, an absolute risk of 12% is low. Inaddition, he knows that based on a CRR of 0.9 and a percentile risk of49^(th) percentile, that the majority of the population is at higherrisk than him. He has confidence in a stable result such as the geneticprofile as compared to PSA, which can fluctuate due to other, benigncauses. Furthermore, the PSA cut-point of 4.0 ng/ml is a borderlineresult; addition of genetic information provided further guidance toallow for a meaningful decision.

Example 3 Patient C

Patient A&B's 60 year old brother heard from his brother about theabove-described genetic test for PCa risk. He has been seeing aurologist for five years regarding an elevation in his PSA and he had anegative biopsy two years ago. His PSA is continuing to climb and he andhis urologist are considering a repeat biopsy. He sees his urologist toconsider what additional information this new genetic test may offer.Together he and his urologist decide that he will have this genetic testdone. His nucleic acid sample, along with his clinical information (age,family history, PSA, F/T PSA ratio, prostate volume from his last biopsyand number of negative cores at the time of his biopsy) are sent in foranalysis. He returns to his urologist's office for the results of histests, which are as follows.

-   -   CRR: 2.52    -   Percentile risk: 95^(th) percentile    -   Absolute risk: 0.32    -   PCa risk score: 0.37

Given the above profile information, Patient C decides to undergo arepeat prostate biopsy, which is positive for Gleason 3+4 PCa. Heundergoes a radical prostatectomy and is cured. Provision of his geneticrisk allowed for the patient to be able to have an outside objectiveassessment of his risk, which is apart from the currently availablepredictors which were abnormal and prompted the initial biopsy. Forpatient C, with a 2.5 fold higher risk with only 5% of the population athigher risk and an absolute risk of 32%, in his opinion, especiallygiven his family history, he wished to pursue repeat biopsy.

Example 4 Methods of Genetic Test to Determine Genetic Score

In a hierarchical order, three models were used to predict PCa risk.First, we used a “genetic marker only” model in which 33 SNPs identifiedby genome wide association studies (GWAS) as associated with PCa riskwere included. Second, we used a “genetic marker+pre-biopsy variablemodel”; in addition to the 33 SNPs, this model included age, familyhistory, and ratio of baseline free PSA to baseline total PSA. Third, weused a “genetic+pre-biopsy variable+post-biopsy variable model”; inaddition to the second model, this model included baseline prostatevolume and number of previous biopsy cores. We used each model toperform risk assessment, which included estimating various measures ofPCa risk, including the cumulative relative risk (CRR), percentile risk,absolute risk, and risk score (i.e., the predicted probability of beingdiagnosed with PCa as determined by a regression model). The predictiveperformance of each model is measured by detection rate of PCa duringthe four years of the REDUCE trial, providing an overall assessment ofclinical validity. Detailed methods for estimating these measures ofrisk are described below.

Odds Ratio (OR) Calculations.

ORs for the 33 SNPs were calculated using external data presented in theliterature. ORs for the clinical variables were estimated from the studysample. For the allelic ORs, we obtained the best estimates and theirconfidence intervals (CIs) for the 33 SNPs using meta-analysis. Thedetails of the meta-analysis are described below. First, if theliterature search yielded raw data such as allele counts of case andcontrol, then we used this information for calculating the OR andstandard error for each study population. Otherwise, we calculated theseestimates using the reported OR and 95% CI. The results from bothapproaches are statistically comparable. Second, while integratingdifferent study results, we began by assessing the heterogeneity ofestimated ORs across study populations. The Q-statistic (for test ofheterogeneity) and 12 statistic (which measures the proportion of totalvariance in estimated ORs due to heterogeneity) were used. If there wasevidence of a high degree of heterogeneity, such as a value of the 12statistic greater than 50%, then the random effects method was used tocalculate the pooled OR and CI. Otherwise, the fixed effects method wasused. The fixed effects method weighs each study with the inverse ofvariance of logarithm of OR, while the random effects methodadditionally incorporates variance in that weight. Furthermore, the ORsfor the demographic and clinical variables were calculated by applyingthe multiple logistic regression in our own study sample since they werenot available from the meta-analysis. Each of the demographic orclinical variables has been categorized with meaningful cut-off points.

Relative Risk (RR) Calculation.

For each of the three genotypes at each SNP, the allelic OR wasconverted to the RR relative to the general population using thefollowing approach. The average population risk compared to non-carrierswas a weighted average of the relative risks of the genotypes.Specifically, the ratio between the average population risk and the riskof non-carriers was estimated by A=P(rr)×OR²+P(wr)×OR+P(ww), where w isthe wild type allele, r is the risk allele, and P(ww), P (wr), and P(rr)are the proportions of the population carrying ww, wr, and rr,respectively. RRs for ww, wr, and rr were estimated by 1/A, OR/A, andOR²/A, respectively. The corresponding confidence intervals wereestimated accounting for variability of estimates of OR. Furthermore,the RRs for the clinical variables were calculated in the similarmanner. The ratio between the average population risk and the risk ofthe reference group was estimated by summing over the product offrequency of each category and the corresponding OR. Then the RR wascalculated accordingly.

Measures of Risk.

Cumulative relative risk (CRR), percentile risk to PCa, absolute risk,and risk score were used as measures of risk to PCa in this study. Toestimate cumulative relative risk, we assumed the controls were a randomsample from the general population. For the genetic only model, amultiplicative model was used, in which we multiplied the RRs for eachof the SNPs for a given individual. For the other two models, the CRRrelative to the population was derived by combining the RRs for the 33SNPs as well as RRs for the clinical variables of the individual bysimple multiplication. The percentile risk to PCa was generated bydetermining the risk level in terms of percentile within thedistribution of population CRR.

The absolute risk for each individual was then estimated based on theoverall CRR, relative to the population (r(a,x)), the incidence rate ofPCa in the general population (λ₀(x)), and the all-cause mortality rateexcluding PCa in the United States (μ₀(x)). Specifically, assuming themortality data are known without error and do not vary with the riskfactors in our model, we used mortality data from the National Center ofHealth Statistics to estimate the mortality rate from non-PCa causes.Let F(a,t) denote the probability that one survives until age t withoutdeveloping PCa. Then F(a,t)=exp{−∫_(a) ^(t)[r(a,x)π₀(x)+μ₀(x)]dx}. Theprobability that one develops PCa in a small interval equals theprobability of his/her disease free survival until age t times theconditional probability of developing PCa by age t+Δt given that one wasdisease free at age t. This probability, absolute risk, is conditionedon the fact that one has not developed PCa by age a. The correspondingCIs can be calculated accounting for the variability of estimates ofrelative risks and of risk factor distributions.

The risk score was the predicted value of PCa risk from a logisticregression model with the CRR from the genetic component alone or incombination with other clinical variables as the covariate. It iscalculated as

$\frac{\exp \left( {{\hat{\beta}}_{0} + {{\hat{\beta}}_{1}X}} \right)}{1 + {\exp \left( {{\hat{\beta}}_{0} + {{\hat{\beta}}_{1}X}} \right)}},$

where X is the relative risk, {circumflex over (β)}₀ and {circumflexover (β)}₁ are regression coefficient estimates for the intercept andrelative risk, respectively. The corresponding CI can be calculated byconverting the CIs for the linear combination of the estimatedcoefficients and the values of the relative risk (i.e., {circumflex over(β)}₀+{circumflex over (β)}₁X).

The distributions of risk score among the REDUCE study subjects arepresented in FIGS. 6 a-c for genetic marker only, geneticmarker+pre-biopsy variable model,” and “genetic+pre-biopsyvariable+post-biopsy variable model,” respectively.

Detection Rate.

In order to assess clinical validity, the detection rate of PCa duringthe 4-year study of the REDUCE study was calculated for each model tomeasure their predictive performance. We divided the sample equally intoquartiles based on the estimated risk of risk. Detection rate was thencalculated as the proportion of positive biopsies in each quartile. Toobtain unbiased estimates, four-fold cross-validation was used tocalculate detection rates. Four-fold cross validation randomly dividesthe data into four (roughly) equal subsets and repeatedly uses threesubsets for model fitting (training) and the remaining subset forvalidation (testing), in order to calculate the detection rate. Thisprocess was repeated until each of the four subsets had been usedexactly once as validation data, after which detection rates wereaveraged across results from each of the 4 validation sets. All of thedetection rates in the testing samples of four-fold cross validationwere reported except for the genetic model, because the genetic scorewas calculated based on external OR estimates of the 33 SNPs. Theobserved detection rates of PCa during the four-year REDUCE study arepresented in FIG. 6 d-f for men at each quartile of estimated risk basedon genetic marker only, genetic marker+pre-biopsy variable model,” and“genetic+pre-biopsy variable+post-biopsy variable model,” respectively.

In some embodiments of this invention, a genetic score that places anindividual in the 50th percentile or greater is indicative of increasedrisk of PCa. An absolute risk value of greater than about 0.13 isindicative of increased risk of PCa. A CRR of greater than 1.0 isindicative of increased risk of PCa. A genetic score that places anindividual below the 50th percentile is indicative of decreased risk ofPCa. An absolute risk value of less than about 0.13 is indicative ofdecreased risk of PCa. A CRR of less than 1.0 is indicative of decreasedrisk of PCa. Increased risk and decreased risk as used herein meanincreased or decreased relative to the general population (see, e.g.,SEER information at http://seer.cancer.gov).

Furthermore, a population median risk score can be used as the cutofffor indicating increased or decreased risk (i.e., a risk score above thecutoff indicates increased risk and a risk score below the cutoffindicates decreased risk). This differs for each of the three models.For genetic only model, the cutoff is 0.24, for genetic+pre-biopsymodel, the cutoff is 0.23 and for genetic+pre-biopsy+post-biopsy, thecutoff is 0.23.

Increased risk and decreased risk as used herein mean increased ordecreased relative to the general population.

Example 5 Clinical Utility of Inherited Markers in Determining Need forRepeat Biopsy: Results from Placebo Arm of the Reduce® Study (Abstract)

Purpose. Management of men following negative prostate biopsy forprostate cancer is challenging. The predictive performance of currentlyavailable clinical parameters such as prostate specific antigen (PSA)for prostate cancer is limited. Recently, 33 PCa risk-associated singlenucleotide polymorphisms (SNPs) have been identified from genome-wideassociation studies. The present study provides an assessment ofsupplementing existing predictors with the prediction of prostate canceron subsequent biopsy.

Methods.

Study subjects included 1,654 men in the placebo arm of the four-yearrandomized REduction by DUtasteride of prostate Cancer Events (REDUCE®)trial, where all subjects had PSAs between 2.5-10.0 ng/mL, a negativeprostate biopsy at baseline and underwent scheduled prostate biopsies atyears 2 and 4.

Results.

Of 1,654 men who had at least one prostate biopsy over four years, 410(25%) and 124 (7%) were diagnosed with prostate cancer and high-gradePCa (Gleason grade≧7), respectively. Differences in the genetic scorebetween men with positive and negative biopsies were highly significanteven after adjusting for other clinical variables (P=3.58×10⁻⁸). The AUCfor prostate cancer prediction of the genetic score was 0.59, higherthan any other individual clinical parameters including PSA (AUC=0.54).When the genetic score was added to the best clinical model includingfive parameters (age, family history, free/total PSA ratio, prostatevolume, and number of cores at base biopsy), the AUC increased from 0.60to 0.64. The differences in detection rates between men with lower orhigher genetic risk at each quartile of estimated risk based on the bestclinical model ranged from 9.31% to 13.66% for prostate cancer and 2.89to 6.16% for high-grade prostate cancer, providing strong evidence forthe added value of genetic markers in risk prediction.

Conclusions.

For men with an initial negative biopsy, genetic markers may be used tosupplement existing predictors to better predict for prostate cancer andhigh-grade prostate cancer on subsequent biopsy.

Example 6 Clinical Utility of Inherited Genetic Markers for thePrediction of Prostate Cancer at Repeat Biopsy: Results from Placebo Armof the Reduce Clinical Trial (Manuscript)

Background.

The predictive performance of available clinical parameters for prostatecancer (PCa) is limited, particularly following negative prostatebiopsy. We sought to assess the clinical utility of identified PCarisk-associated single nucleotide polymorphisms (SNPs) for PCaprediction in a clinical trial.

Methods.

Subjects included 1,654 men who consented for genetic studies in theplacebo arm of the randomized REduction by DUtasteride of ProstateCancer Events (REDUCE) trial, where all subjects had a negative prostatebiopsy at baseline and underwent scheduled prostate biopsies at years 2and 4. Predictive performance of clinical parameters at baseline, and/ora genetic score based on 33 PCa risk-associated SNPs was evaluated usingthe area under the receiver operating characteristic curve (AUC) and PCadetection rate.

Findings.

Of the 1,654 men, 410 (25%) were diagnosed with PCa during the four yearfollow-up. The genetic score based on the 33 SNPs was a highlysignificant predictor for positive biopsy even after adjusting for knownclinical variables (P=3.58×10⁻⁸). Measured by AUC, the genetic scoreoutperformed any individual clinical parameter includingprostate-specific antigen (PSA) for PCa risk prediction, and improvedthe performance of the best combined clinical model consisting of age,family history, free/total PSA ratio, prostate volume, and number ofinitial biopsy cores. The added value of the genetic score ishighlighted by its ability to further differentiate PCa detection ratesdefined by the best clinical model. The observed PCa detection rate over4-years was 19.16% higher for men with higher estimated clinicalrisk/higher genetic score (34.82%) than with lower estimated clinicalrisk/lower genetic score (15.66%), P=3.3×10⁻¹⁰.

Interpretations.

This clinical trial provides the next level of evidence, that germlinemarkers may be used to supplement existing clinical parameters to betterpredict outcome of prostate biopsy.

Introduction.

Prostate cancer (PCa) is the most common solid organ malignancyaffecting American men and the second leading cause of cancer relateddeath.¹ Approximately one million prostate biopsies are performed yearlyin the U.S. The vast majority of these biopsies are performed due toelevated levels of the PCa marker prostate-specific antigen (PSA).However, only a quarter of these biopsies result in a diagnosis of PCa,highlighting the inadequate performance of PSA to predict PCa.Persistently elevated PSA levels and/or other clinical parameters thatprompted initial biopsies contribute to stress and anxiety among bothpatients and their urologists.² Novel biomarkers are urgently needed tobetter determine the need for initial and repeat prostate biopsy.

Recently, more than 30 PCa risk-associated single nucleotidepolymorphisms (SNPs) have been discovered from genome-wide associationstudies (GWAS).³⁻¹³ These risk-associated SNPs have been consistentlyreplicated in multiple case-control study populations of Europeandescent.¹⁴ Although each of these SNPs is only moderately associatedwith PCa risk, a genetic score based on a combination of risk-associatedSNPs can be used to identify men at high risk for PCa.¹⁵⁻¹⁸ Theserisk-associated SNPs may have broad practical applications because theyare common in the general population.

Study Population.

Subjects included 1,654 of the 3,129 (53%) men of European descent inthe placebo arm of the randomized, multi-institutional, international,Reduction by DUtasteride of Prostate Cancer Events (REDUCE) study whoconsented for genetic studies. The characteristics of patients whoconsented or declined genetic studies are presented in Table 3. TheREDUCE study is a randomized double blind chemoprevention trial,examining PCa risk reduction by dutasteride, a dual 5-alpha reductaseinhibitor, in a population of men with prior negative prostate biopsy.¹⁹Eligible men were 50 to 75 years of age, with a serum PSA≧2.5 ng/mL and≦10 ng/mL (men aged 50-60 years) or ≧3.0 ng/mL and ≦10 ng/mL (men>60years of age), and had a single, negative prostate biopsy (6-12 cores)within 6 months prior to enrollment (independent of the study).Exclusion criteria included more than one prior prostate biopsy,high-grade prostatic intra-epithelial neoplasia (HG-PIN) or atypicalsmall acinar proliferation (ASAP) on the pre-study entry prostate biopsyassessed by a central pathology laboratory, or a prostate volume greaterthan 80 cc.

PCa Risk-Associated SNPs, Ancestry Informative Markers (AIMs), andGenotyping.

A panel of 33 PCa risk-associated SNPs were selected from all PCa GWASreported before December 2009 (Table 4). Each of these SNPs exceededgenome-wide significance levels in their initial reports (P<10⁻⁷) andthese associations have been replicated in independent studypopulations.³⁻¹³ In addition, 91 SNPs from a panel of 93 AIMs weregenotyped to distinguish population groups from major continents.²⁰These SNPs were genotyped using the Sequenom MassARRAY platform. Oneduplicated CEPH (Centre d'Etude du Polymorphisme Humain) sample and twowater samples (negative controls) that were blinded to technicians wereincluded in each 96-well plate. The concordance rate between the twogenotype calls of the duplicated CEPH sample for all SNPs was 100%.

Statistical Analyses.

Allelic odds ratios (ORs) and 95% confidence intervals (CIs) for each ofthe 33 SNPs were estimated using an unconditional logistic regressionmodel, adjusting for ethnic structure using the first two principalcomponents, as is standard in genetic association studies.²⁰⁻²¹ (Table4). A genetic score, based on all 33 SNPs and OR estimates from anexternal meta-analysis, was calculated for each individual.²² Briefly, amultiplicative model was used to derive genotype relative risks from theexternal allelic OR. For each of the three genotypes at each SNP, thegenotype relative risk was converted to the risk, relative to thepopulation. The overall risk, relative to the population (i.e., geneticscore), was derived by combining the risks, relative to the population,of all SNPs of each individual by simple multiplication.

Chi-square and t-tests were used to compare the differences betweengroups of subjects for binary variables (family history, digital rectalexam [DRE], and continuous variables (age, PSA measurements, prostatevolume, number of cores at pre-study entry biopsy, and genetic score),respectively. Total PSA and genetic score were log transformed toapproach a normal distribution.

The AUC of clinical predictors and genetic score, individually and incombination, for predicting PCa was estimated using a logisticregression model. Four-fold cross validation was used to reduce the biasin estimates of AUC. Subjects were randomly divided into four groups. Amodel was fit to each three-quarter subset of the subjects and tested onthe remaining one-quarter subset of subjects, yielding four testingAUCs. Results from 10 runs of four-fold cross validation are reported.

We also calculated the detection rate of PCa for men at variousestimated risk categories based on prediction models. Unbiased detectionrates were directly estimated for the genetic model, because the geneticscore of each individual was calculated based on external OR estimatesof the 33 SNPs. For the clinical model, four-fold cross validation wasused to obtain unbiased estimates, as described below. Coefficients ofvariables in the prediction models were estimated from eachthree-quarter subset of the subjects and used to calculate risk in theremaining one-quarter subset of subjects. Each of these one-quartersubsets of subjects was ranked based on estimated risk and then equallydivided into two groups. The PCa detection rate was calculated as theproportion of positive biopsy in each group. Results from 10 runs offour-fold cross validation are reported.

Results.

Among the 1,654 men of European descent who had an initial negativebiopsy for PCa and who consented to genetic studies in the placebo armof the REDUCE trial, 410 men (25%) had a positive prostate biopsy forPCa from scheduled and for-cause biopsies over the four-year study. In aunivariate analysis (Table 1), men with positive biopsies differedsignificantly (P<0.05) from men with negative prostate biopsies for allof the baseline clinical and demographic variables, with the exceptionof DRE. Significant differences were also observed for genetic riskfactors; positive family history of PCa was found in 17% of the men withpositive biopsy, compared with 12% of the men with negative biopsy(OR=1.5 [95% CI: 1.09-2.04], P=0.01), and the difference in the geneticscore between these two groups was highly significant (P=4.95×10⁻⁹).After adjusting for known PCa risk-associated clinical variables such asage, free/total PSA ratio, number of cores at initial biopsy, andprostate volume using multivariate logistic regression analysis, familyhistory and genetic score remained significantly associated withpositive prostate biopsy (P=0.002 and 3.58×10⁻⁸, respectively).

We calculated the AUC of these baseline clinical variables and geneticrisk factors, individually and in combination, for predicting positiveprostate biopsy during the four-year follow-up. To obtain unbiasedestimates of AUC, a four-fold cross validation method was used andresults from testing samples are reported (Table 2). Among individualpredictors, the AUC of the genetic score was highest (0.59), followed byprostate volume (0.56), age (0.56), number of cores sampled at pre-studyentry biopsy (0.55), free/total PSA ratio (0.54), total PSA (0.54),family history (0.52), and DRE (0.51). When multiple predictors wereincluded in the model simultaneously, the best clinical model includedfive baseline variables (age, family history, free/total PSA ratio,number of cores at pre-study entry biopsy, and prostate volume), with anAUC of 0.60. When the genetic score was added to this best clinicalmodel, the AUC increased to 0.64.

To facilitate the use and interpretation of these models in predictingpositive prostate biopsy, we calculated the PCa detection rate duringfour years for the genetic score model and the best clinical model. Eachindividual's risk for PCa was estimated using either the genetic scoremodel or the best clinical model, and was classified as being lower orhigher risk for PCa (compared to the median risk) under each model. Theobserved detection rates of PCa for men at different estimated risksunder each model are presented in FIGS. 1 a-b. Both the genetic modeland the best clinical model were able to differentiate detection ratebetween these two groups of men, although the genetic model performedbetter. In the genetic model, the observed detection rate was 11.60%higher for men who had higher estimated risk (30.59%) than those withlower estimated risk (18.99%). The difference was highly significant,P=4.6×10⁻⁸. In the best clinical model, the observed detection rate was8.65% higher for men who had higher estimated risk (29.16%) than thosewith lower estimated risk (20.51%). The difference was also significant,P=5.4×10⁻⁵.

To further examine the value of adding the genetic score to existingclinical parameters in predicting positive prostate biopsy, we estimatedPCa detection rates among men who were classified as the same risk basedon the best clinical model but having different genetic scores (FIG. 2).The genetic score was able to further differentiate detection rate. Formen at lower clinical risk, the detection rate for PCa was 9.90% higherfor men whose genetic score was above the median (25.56%) than thosebelow the median (15.66%), P=4.9×10⁻⁴. Similarly, for men at higherclinical risk, the detection rate for PCa was 11.48% higher for men whohad higher genetic score (34.82%) than lower genetic score (23.34%),P=3.2×10⁻⁴. Combining the genetic model and the best clinical model,they were able to considerably differentiate detection rate between theextreme groups of men. The detection rate was 19.16% higher for men whohave higher estimated clinical risk/higher genetic score (34.82%) thanmen who had lower estimated clinical risk/lower genetic score (15.66%),P=3.3×10⁻¹⁰.

To preliminarily evaluate the performance of genetic score and clinicalparameters in distinguishing risk for high-grade PCa, we compared thedetection rate of high-grade PCa among men with various estimated riskunder these two models. Among the 410 men who were diagnosed with PCa,124 (30%) had high-grade PCa (Gleason grade≧7). Higher detection rateswere observed among men with higher estimated risk compared to thosewith lower risk under the genetic model (FIG. 3 a), the best clinicalmodel (FIG. 3 b), and the combination of both models (FIG. 3 c).

Results from several retrospective case-control studies have suggestedthat PCa risk-associated SNPs discovered from GWAS may be used topredict an individual's risk for PCa, providing the possibility thatthey may be used for targeted screening and chemo-prevention ofPCa.¹⁵⁻¹⁶ However, due to limitations of the case-control study design,a number of key questions have remained prior to their clinical use. Thefirst fundamental question is whether these SNPs are associated withelevated PSA and not PCa risk per se, as elevated PSA leads to moreprostate biopsies and in turn a greater PCa detection rate as is seen incase control studies (i.e., PSA detection bias).²³ Another importantquestion is the assessment of predictive performance of genetic markersand clinical variables such as PSA in the same study, and moreimportantly whether genetic markers significantly improve the ability ofexisting clinical parameters to predict PCa. These questions aredifficult to address in case-control studies as these clinical variablesare commonly used as part of PCa screening.

The placebo arm of the REDUCE study, a large randomized clinical trial,provides a unique opportunity to answer these two important questions.All men in the study had a negative biopsy at baseline and werefollowed-up for four years, with scheduled not-for-cause (i.e.,regardless of PSA levels and other clinical indications) prostatebiopsies at years 2 and 4. Therefore, this study design minimizes thepotential impact of PSA detection bias on associations between PCa riskand SNPs. In addition, because it is a clinical trial, a number ofclinical variables, such as free/total PSA ratio and prostate volumewere measured at baseline using a standardized protocol. To ourknowledge, this is the first reported study to validate these PCarisk-associated SNPs and assess their value when added to existingclinical variables for the prediction of PCa risk in a large prospectiveclinical trial. In this study, we found that the genetic score is asignificant predictor of positive prostate biopsy and that thisassociation is independent of known clinical parameters and familyhistory (P=3.58×10⁻⁸). Considering that the genetic score was based onall 33 a priori established PCa risk-associated SNPs and using ORestimates obtained from external study populations, these resultsprovide the highest level of independent evidence of the validity ofthese genetic markers to predict an individual's risk for PCa. Inaddition, through a direct comparison of the predictive performance(AUC) of genetic markers and existing clinical variables in the samestudy population, we showed that the genetic score outperformed anyother individual clinical parameter, including PSA, for PCa riskprediction. More importantly, the genetic score improved the AUC whenadded to a model including the best, existing clinical variables.

The strongest support for the predictive performance of genetic markersand added value of genetic markers to the existing clinical variables inthis population is demonstrated by the measurement of detection rate ofPCa. The ˜10% difference in detection rate of PCa between higher orlower genetic score and ˜20% difference between the two extreme groups(men with lower clinical risk and lower genetic score, or higherclinical risk and higher genetic score) may be clinically significant.This improvement is worth noting considering that few other biomarkersin the past several decades, be they proteins or genetic markers, havereached such a level. It is also important to note that detection rate,as a measurement of predictive performance, can be easily understood andinterpreted by physicians and patients. This is in contrast to AUC,another commonly used measurement of predictive performance, where thevalue is not directly related to meaningful clinical measurements.

There are fundamental differences between the genetic score and clinicalvariables. An advantage of clinical variables is that they directlyassess parameters that are associated with the development of thedisease. On the other hand, the genetic score assesses the likelihood ofdeveloping disease and thus is time-independent. It can be assessed atany stage, before or after the development of disease. The highstability of DNA molecules as well as accurate and low cost genotypingof genetic markers also facilitates their clinical implementation. Somepotential applications of genetic markers may include the identificationof high risk men at a younger age for PCa screening and chemoprevention,as well as supplementation of the clinical variables to determine theneed for biopsy or, as in this study, the need for repeat biopsy.

Results from this study not only add further support for the utility ofgenetic markers in predicting PCa risk but also provide new informationthat is urgently needed for the management of the ˜750,000 American menyearly who have a negative prostate biopsy. Currently, PSA levels andfree/total PSA ratio are the primary predictors used to determine theneed and interval for repeat prostate biopsy.² Their ability to predictPCa is unsatisfactory, with published AUCs in the 0.60-0.75 range.²⁴⁻²⁶The predictive performance of PSA was even lower in our study, with anAUC of 0.54 for total PSA or free/total PSA ratio. The lower AUCestimate in our study may be due to the repeat biopsy population or thefewer PSA-driven biopsies (less than 7% PCa were detected byprotocol-independent biopsies).¹⁹ In addition, the AUCs reported in ourstudy were based on testing samples of four-fold cross-validation, whichminimizes the upward bias due to model over-fitting. Regardless of thedifferent estimates of AUC from different studies, the generally low AUCin all of the studies points to the need for additional markers tobetter guide indications for repeat biopsy and determine the timing offollow-up. To this end, this study has successfully demonstrated that agenetic score based on PCa risk-associated SNPs may be one of these muchneeded markers.

There are several notable limitations in this study. One of the mostimportant drawbacks was that the study was limited to subjects ofEuropean descent. This is in part due to the fact that PCarisk-associated SNPs were discovered in men of European descent. Therelevance of these SNPs in other races is unknown, although PCaassociations with several of these risk-associated SNPs have beenconfirmed in men of African American, Asian, and Hispanic race.²⁷Furthermore, only a small number of men of non-European descentparticipated in the REDUCE trial,¹⁹ thus significantly limiting thepower to draw any conclusions beyond this one ethnicity. Anotherimportant limitation was that we did not directly assess the ability ofthese genetic markers to independently discriminate risk betweenhigh-grade and low-grade PCa, although we have demonstrated the addedvalue of the genetic score for predicting high-grade PCa by detectionrate. Several studies have previously suggested that these 33 SNPs arenot able to distinguish risk for aggressive PCa from its more indolentform.²⁸⁻²⁹ In addition, due to the relatively low frequency ofhigh-grade PCa patients in this study, the statistical power is limited.Finally, it is important to note that the predictive performance of thebest clinical model and genetic model remain poor.

Our study validated the association of a genetic score based on 33 SNPswith PCa risk in the context of a prospective clinical trial, and forthe first time, demonstrated the added value of genetic markers to theexisting clinical variables for PCa prediction. The improvement ofgenetic markers in predicting PCa, albeit moderate, is much needed forurologists and their patients to determine the need for biopsy, and inparticular repeat biopsy, for PCa detection.

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N Engl J Med. 2008; 358:910-919.-   16. Xu J, Sun J, Kader A K, Lindstrom S, Wiklund F, Hsu F C, et al.    Estimation of absolute risk for prostate cancer using genetic    markers and family history. Prostate. 2009; 69(14):1565-1572.-   17. Salinas C A, Koopmeiners J S, Kwon E M, FitzGerald L, Lin D W,    Ostrander E A, et al. Clinical utility of five genetic variants for    predicting prostate cancer risk and mortality. Prostate. 2009;    69(4):363-372.-   18. Fitzgerald L M, Kwon E M, Koopmeiners J S, Salinas C A, Stanford    J L, Ostrander E A. Analysis of recently identified prostate cancer    susceptibility loci in a population-based study: associations with    family history and clinical features. Clin Cancer Res. 2009;    15:3231-3237.-   19. Andriole G A, Bostwick D, Brawley O W. The influence of    dutasteride on the risk of biopsy-detectable prostate cancer:    Outcomes of the REduction by DUtasteride of Prostate Cancer Events    (REDUCE) study. N Engl J. Med. 2010; 362(13):1192-1202.-   20. Nassir R, Kosoy R, Tian C, White P A, Butler L M, Silva G, et    al. An ancestry informative marker set for determining continental    origin: validation and extension using human genome diversity    panels. BMC Genet. 2009; 10:39.-   21. Price A L, Patterson N J, Plenge R M, Weinblatt M E, Shadick N    A, Reich D, et al. Principal components analysis corrects for    stratification in genome-wide association studies. Nat Genet. 2006;    38:904-909.-   22. Kim S-T, Cheng Y, Hsu F-C, Jin T, Kader A K, Zheng S L, et al.    Prostate cancer risk-associated variants reported from genome-wide    association studies: meta-analysis and their contribution to genetic    variation. Prostate. 2010 Jun. 16-   23. Ahn J, Berndt S I, Wacholder S, Kraft P, Kibel A S, Yeager M, et    al. Variation in KLK genes, prostate-specific antigen and risk of    prostate cancer. Nat Genet. 2008; 40:1032-1034.-   24. 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Example 7 Additional Description and Data

Background of the Problem that is Addressed.

Prostate cancer (PCa) is the most common solid organ malignancyaffecting American men and the second leading cause of cancer relateddeath. There are at least two major problems in diagnosing andpreventing PCa: 1) it is difficult to predict men at elevated risk forPCa, and 2) it is difficult to predict outcome of prostate biopsy.

Recently, 33 PCa risk-associated single nucleotide polymorphisms (SNPs)have been identified. We assessed the ability of these 33 inherited PCarisk-associated genetic markers to address the problems listed above.

Brief Summary of the Invention.

Using clinical data and DNA samples from the REduction by DUtasteride ofprostate Cancer Events (REDUCE) trial, we have obtained novel resultsthat may have broad clinical utility:

-   -   a) Genetic score based on a panel of 33 PCa risk-associated SNPs        (PCS33) can predict an individual's risk for PCa.    -   b) Genetic score based on PCS33 can supplement current clinical        variables (PSA, prostate volume, age, and family history) to        better determine the clinical decision to pursue prostate biopsy        (or repeat prostate biopsy) for detection of PCa.

Description of a) Genetic Score Based on a Panel of 33 PCaRisk-Associated SNPs (PC-S33) can Predict Individual Risk for PCa, andb) Genetic Score Based on PC-S33 can Supplement Current ClinicalVariables (PSA, Prostate Volume, Age, and Family History) to BetterDetermine the Clinical Decision to Perform a Prostate Biopsy (or RepeatProstate Biopsy) for PCa Detection.

These were conceived prior to and confirmed using the population in theplacebo arm of the REDUCE study.

Among the 1,654 men of European descent who had an initial negativebiopsy for PCa and who consented to genetic study in the placebo arm ofthe REDUCE trial, 410 men (25%) had a positive prostate biopsy for PCafrom scheduled and for-cause biopsies over the four-year study. In aunivariate analysis (Table 5), men with positive biopsies hadsignificantly higher genetic score based on PCS33 than men with negativeprostate biopsy (P=4.95×10⁻⁹). After adjusting for known PCarisk-associated clinical variables such as age, free/total PSA ratio,number of cores at base biopsy, and prostate volume using multivariatelogistic regression analysis, and family history, the genetic scoreremained significantly associated with positive prostate biopsy(P=3.58×10⁻⁸). The results from this prospective clinical trialestablish the basis for the use of these genetic markers to predict anindividual's risk for PCa.

We used the area under the receiver operating characteristic curve (AUC)to assess the performance of these baseline clinical variables andgenetic score, individually and in combination, to predict for positiveprostate biopsy during the four-year follow-up. To obtain unbiasedestimates of AUC, a four-fold cross validation method was used andresults from testing samples were reported (Table 6). The AUC of thegenetic score was highest (0.59) among individual predictors; includingprostate volume (0.56), age (0.56), number of cores sampled at pre-studyentry biopsy (0.55), free/total PSA ratio (0.54), total PSA (0.54),family history (0.52), and DRE (0.51). When multiple predictors wereincluded in the model simultaneously, the AUC for commonly usedpredictors including age, family history, and total PSA was 0.58. Thebest clinical model included five baseline variables (age, familyhistory, free/total PSA ratio, number of cores at pre-study entrybiopsy, and prostate volume), with an AUC of 0.60. When the geneticscore was added to this best clinical model, the AUC of the full modelincreased to 0.64.

To facilitate the use and interpretation of these models in predictingpositive prostate biopsy, we calculated the detection rate of PCa andhigh-grade PCa for the genetic score model, the best clinical model, andthe full model (FIG. 4). For each model, the detection rate generallyincreased in men with increasingly higher estimated risk. The differencein PCa detection rate between the lowest and highest quartile was14.08%, 11.78%, and 12.14% for the genetic score model, the bestclinical model, and the full model that combined genetic score with thebest clinical model, respectively (FIGS. 4 a-c). The difference inhigh-grade PCa detection rate between the lowest and highest quartilewas 4.37%, 7.03%, and 7.63% for the genetic model, the best clinicalmodel, and the full model, respectively (FIGS. 4 d-f).

To further examine the added value of the genetic score to the existingclinical parameters in predicting positive prostate biopsy, we estimatedPCa detection rates in each quartile of risk based on the best clinicalmodel, stratified by genetic score (lower and higher half) (FIG. 5 a).Within each clinical risk quartile, the detection rates differedconsiderably between men with lower and higher genetic scores; thedifference was 10.38% in the 1st, 9.42% in the 2nd, 13.66% in the 3rd,and 9.31% in the 4th risk quartile, respectively. Comparing across therisk quartiles, men with higher genetic scores, even in the lowerclinical risk quartile, had comparable or even higher PCa detection ratethan men with lower genetic scores in any clinical risk quartile.Specifically, the PCa detection rate was 25.64% for men that had ahigher genetic score within the lowest clinical risk quartile; this iscomparable or higher than the detection rates among men that had a lowergenetic score in the 2nd, 3rd, or highest clinical risk quartile(16.06%, 19.34%, and 27.34%, respectively). Similarly, genetic score wasable to further differentiate the detection rate of high-grade PCadefined by the best clinical model (FIG. 5 b).

Through a direct comparison of the predictive performance (AUC) of thegenetic score and existing clinical variables in the same studypopulation, we showed that the genetic score performed better than anyother individual clinical parameter, including PSA, for PCa riskprediction. More importantly, the genetic score improved the AUC ofexisting clinical variables. The strongest support for the added valueof the genetic score to the existing clinical variables in thispopulation is reflected by the ability of the genetic score todifferentiate PCa detection rates among men in the same risk quartiledefined by the best clinical model.

Prior to our study, it was not known whether reported PCarisk-associated SNPs are false positive due to PSA detection bias (i.e.,these SNPs are associated with elevated PSA and not PCa risk per se, aselevated PSA leads to more prostate biopsies and in turn a greater PCadetection rate as is seen in case control studies). In addition, becausemany clinical variables such as PSA and DRE are commonly used to definecases and controls in case-control studies, it is difficult to assessrelative predictive performance of genetic markers and clinicalvariables such as PSA, and more importantly whether genetic markersconsiderably improve the ability of existing clinical parameters topredict for PCa.

The placebo arm of the REDUCE study, a large randomized clinical trial,provided a unique opportunity to answer these questions. All men in thestudy had a negative biopsy at baseline and were followed-up for fouryears, with scheduled not-for-cause prostate biopsies at years 2 and 4.In addition, because it is a clinical trial, a number of clinicalvariables, such as free/total PSA ratio and prostate volume weremeasured at baseline using a standardized protocol. To our knowledge,our findings were the first to establish the clinical validity of thesePCa risk-associated SNPs and the value they add to existing clinicalvariables for the prediction of PCa risk in a large prospective clinicaltrial.

On the basis of these studies, we have developed a genetic test usingPCS33 to determine the need for prostate biopsy.

Example 8 Analysis of Randomly Selected Subsets of the 33 SNPS Of Table1

Calculations as described herein were performed on 10 and 15 randomlyselected SNPs (Table 8) that are subsets of the 33 SNPs of Table 1 andthis random sampling was repeated five times. The genetic scores (CRRs)calculated from these subsets is equivalent or better that the familyhistory for detecting prostate cancer risk measured by AUC (Table 7).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the claimsprovided herein, with equivalents of the claims to be included therein.

All publications, patent applications, patents, patent publications,sequences identified by GenBank® Database accession numbers and/or SNPaccession numbers, and other references cited herein are incorporated byreference in their entireties for the teachings relevant to the sentenceand/or paragraph in which the reference is presented.

TABLE 1 Reported SNPs associated with PCa and their odds ratio from ameta-analysis m/M* Risk CHR SNPs Note BP-build36 Known genes alleleallele OR (95% CI) 2 rs1465618 2p21 43,407,453 THADA A/G A 1.15(1.04-1.26) 2 rs721048 2p15 62,985,235 EHBP1 A/G A 1.16 (1.11-1.22) 2rs12621278 2q31.1 173,019,799 ITGA6 G/A A 1.35 (1.27-1.44) 3 rs26607533p12 87,193,364 T/C T 1.24 (1.04-1.48) 3 rs10934853 3q21.3 129,521,063A/C A 1.12 (1.06-1.18) 4 rs17021918 4q22.3 95,781,900 PDLIM5 T/C C 1.14(1.10-1.18) 4 rs7679673 4q24 106,280,983 TET2 A/C C 1.13 (1.10-1.17) 6rs9364554 6q25 160,753,654 T/C T 1.17 (1.06-1.29) 7 rs10486567 7p1527,943,088 JAZF1 A/G G 1.16 (1.10-1.23) 7 rs6465657 7q21 97,654,263LMTK2 T/C C 1.14 (1.05-1.23) 8 rs2928679 8p21.2 23,494,920 NKX3.1 A/G A1.13 (1.02-1.25) 8 rs1512268 8p21.2 23,582,408 NKX3.1 T/C T 1.17(1.14-1.21) 8 N10086908 8q24 (5) 128,081,119 C/T T 1.13 (1.09-1.18) 8rs16901979 8q24 (2) 128,194,098 A/C A 1.80 (1.57-2.06) 8 rs169020948q24.21 128,389,528 N/A G 1.20 (1.12-1.30) 8 rs620861 8q24 (4)128,404,855 A/G G 1.16 (1.11-1.20) 8 rs6983267 8q24 (3) 128,482,487 G/TG 1.20 (1.14-1.26) 8 rs1447295 8q24 (1) 128,554,220 A/C A 1.47(1.33-1.62) 9 rs1571801 9q33 123,467,194 G/A A 1.17 (0.95-1.45) 10rs10993994 10q11 51,219,502 MSMB T/C T 1.25 (1.12-1.40) 10 rs496241610q26 126,686,862 CTBP2 C/T C 1.15 (1.04-1.27) 11 rs7127900 11P15.52,190,150 IGF2, IGF2AS, INS, TH G/A A 1.25 (1.20-1.30) 11 rs1241845111q13 (2) 68,691,995 AL137479, BC043531 A/G A 1.16 (1.09-1.23) 11rs10896449 11q13 (1) 68,751,243 A/G G 1.16 (1.11-1.22) 17 rs1164974317q12 (2) 33,149,092 A/G G 1.16 (1.11-1.22) 17 rs4430796 17q12 (1)33,172,153 TCF2 A/G A 1.22 (1.17-1.26) 17 rs1859962 17q24.3 66,620,348G/T G 1.20 (1.13-1.27) 19 rs8102476 19q13.2 43,427,453 T/C C 1.12(1.08-1.15) 19 rs887391 19q13 46,677,464 10 Mb to KLK3 C/T T 1.14(1.08-1.20) 19 rs2735839 19q13 (KLK3) 56,056,435 KLK3 A/G G 1.30(1.11-1.51) 22 rs9623117 22q13 38,782,065 C/T C 1.13 (1.05-1.22) 22rs5759167 22q13.2 41,830,156 TTLL1, BIK, MCAT, PACSIN2 T/G G 1.18(1.14-1.21) 23 rs5945619 Xp11 51,258,412 NUDT10, NUDT11, LOC340602 C/T C1.27 (1.12-1.43) *m = minor allele, M = major allele.

TABLE 2 Clinical and genetic predictors of prostate cancer Testing AUCfrom four- fold cross Variables and models validation Individualvariables at baseline Age at baseline (Age) 0.56 Digital rectalexamination at baseline (DRE) 0.51 Total PSA levels at baseline 0.54Free/total PSA ratio at baseline (f/t PSA) 0.54 Prostate volume atbaseline (PV) 0.56 Number of cores sampled at base biopsy (No. of cores)0.55 Family history at baseline (FH) 0.53 Genetic score based on 33 PCarisk SNPs (Genetic score) 0.59 Combined variables Age + FH + total PSA0.58 Age + FH + f/t PSA 0.59 Age + FH + DRE + f/t PSA 0.59 Age + FH +f/t PSA + PV + No. of cores 0.60 Age + FH + f/t PSA + PV + No. ofcores + Genetic score 0.64

TABLE 3 Baseline clinical, demographic, and genetic score of thesubjects in the study All subjects Positive Negative Variables BiopsiesBiopsies P-values Number of subjects 410 1244 Age at baseline Mean (SD),years 63.52 (5.99)  62.22 (6.01)  0.0001 Range 50-76 49-76 # (%) withpositive    68 (17%)  146 (12%) 0.01 family history at baseline # (%)with positive    20 (5%)  47 (4%) 0.33 DRE^(†) at baseline Total PSAlevels at baseline Mean (SD), mL 5.78 (1.37) 5.52 (1.40) 0.01 Range, mL 2.5-10.2  1.8-14.2 Free/total PSA ratio 0.16 (0.06) 0.17 (0.06) 0.02 atbaseline Prostate volume at baseline 44.20 (21.40) 46.76 (16.13) 0.03Number of cores sampled 8.21 (2.27) 8.58 (2.39) 0.004 at base biopsyGenetic score based on 0.94 (1.83) 0.77 (1.81) 4.95E−09 33 PCa risk SNPs^(†)DRE: Digital rectal examination

TABLE 4 Comparison of characteristics for men in the placebo groupconsented or declined genetic studies Consented for genetic studiesVariables Yes No P-values Number of subjects 1654 1475 Age at baselineMean (SD), years 62.55 (6.03)  62.87 (6.03)  0.13 Range 49-76 49-77 #(%) with positive family   214 (12.94)   188 (12.75) 0.87 history atbaseline # (%) with positive   67 (4.06)   51 (3.47) 0.39 DRE^(†) atbaseline Total PSA levels at baseline Mean (SD), ml 5.89 (1.89) 5.98(1.97) 0.18 Range, mL  1.8-14.2  2.4-23.2 Free/total PSA ratio atbaseline Mean (SD), mL 0.16 (0.06) 0.17 (0.06) 0.02 Range, mL 0.03-0.470.04-0.64 Prostate volume at baseline Mean (SD), mL 46.13 (17.62) 44.58(17.61) 0.02 Range, mL  3.66-256.83  5.75-264.94 ^(†)DRE: Digital rectalexamination

TABLE 5 Baseline clinical, demographic, and genetic score of thesubjects in the study All subjects Subjects with positive BiopsiesVariables Positive Biopsies Negative Biopsies P-values Gleason grade ≦6Gleason grade ≧7 P-values Number of subjects 410 1244 286 124 Age atbaseline Mean (SD), years 63.52 (5.99)  62.22 (6.01)  0.0001 63.01(6.02)  64.72 (5.75) 0.008 Range 50-76 49-76 50-76 52-75 # (%) withpositive family history  68 (17%) 146 (12%) 0.01  44 (15%)  24 (19%)0.32 at baseline # (%) with positive DRE^(†) at 20 (5%) 47 (4%) 0.33 15(5%)  5 (4%) 0.60 baseline Total PSA levels at baseline Mean (SD), mL5.78 (1.37) 5.52 (1.40) 0.01 5.62 (1.37) 6.16 (1.36) 0.008 Range, mL 2.5-10.2  1.8-14.2  2.5-10.2 2.7-10  Free/total PSA ratio at baseline0.16 (0.06) 0.17 (0.06) 0.02 0.16 (0.06) 0.15 (0.07) 0.32 Prostatevolume at baseline 44.20 (21.40) 46.76 (16.13) 0.03 45.29 (22.54) 41.72(18.38) 0.10 Number of cores sampled at base 8.21 (2.27) 8.58 (2.39)0.004 8.30 (2.15) 8.00 (2.51) 0.09 biopsy Genetic score based on 33 PCa0.94 (1.83) 0.77 (1.81) 4.95E−09 0.93 (1.84) 0.96 (1.80) 0.66 risk SNPs^(†)DRE: Digital rectal examination

TABLE 6 Clinical and genetic predictors of prostate cancer andhigh-grade prostate cancer Testing AUC from four-fold cross validationHigh-grade Any prostate prostate Variables and models cancer cancerIndividual variables at baseline Age at baseline (Age) 0.56 0.61 Digitalrectal examination at baseline (DRE) 0.51 0.50 Total PSA levels atbaseline 0.54 0.59 Free/total PSA ratio at baseline (f/t PSA) 0.54 0.57Prostate volume at baseline (PV) 0.56 0.59 Number of cores sampled atbase biopsy 0.55 0.58 (No. of cores) Family history at baseline (FH)0.53 0.54 Genetic score based on 33 PCa risk SNPs 0.59 0.57 (Geneticscore) Combined variables Age + FH + total PSA 0.58 0.65 Age + FH + f/tPSA 0.59 0.65 Age + FH + DRE + f/t PSA 0.59 0.65 Age + FH + f/t PSA +PV + No. of cores 0.60 0.67 Age + FH + f/t PSA + PV + No. of 0.64 0.67cores + Genetic score High-grade prostate cancer is defined as Gleasongrade 7 or higher

TABLE 7 Random Random Random Random Random sample 1 sample 2 sample 3sample 4 sample 5 FH 0.53 GS33 0.59 GS15 0.56 0.55 0.56 0.53 0.55 GS100.54 0.54 0.54 0.56 0.53

TABLE 8 Random Random Random Random Random Sample 1 Sample 2 Sample 3Sample 4 Sample 5 15 SNPs rs1465618 rs1465618 rs10934853 rs721048rs1465618 rs12621278 rs721048 rs17021918 rs12621278 rs721048 rs7679673rs12621278 rs10486567 rs2660753 rs12621278 rs6465657 rs17021918rs6465657 rs17021918 rs10934853 rs2928679 rs7679673 rs2928679 rs7679673rs2928679 rs1512268 rs9364554 rs1512268 rs9364554 rs10086908 rs16901979rs6465657 rs16901979 rs10486567 rs620861 rs620861 rs10086908 rs620861rs2928679 rs7127900 rs10993994 rs16902094 rs10993994 rs10086908rs12418451 rs7127900 rs620861 rs7127900 rs6983267 rs11649743 rs12418451rs11649743 rs12418451 rs4962416 rs4430796 rs11649743 rs1859962 rs8102476rs7127900 rs1859962 rs4430796 rs2735839 rs887391 rs12418451 rs8102476rs2735839 rs9623117 rs9623117 rs11649743 rs9623117 rs5945619 rs5759167rs5945619 rs887391 rs5759167 10 SNPs rs17021918 rs1465618 rs1465618rs1465618 rs1465618 rs9364554 rs12621278 rs12621278 rs10934853rs12621278 rs6465657 rs10934853 rs1512268 rs6465657 rs7679673 rs2928679rs9364554 rs16901979 rs2928679 rs6465657 rs1512268 rs10486567 rs1571801rs1512268 rs1571801 rs16901979 rs10086908 rs10993994 rs10086908rs7127900 rs1571801 rs620861 rs12418451 rs6983267 rs8102476 rs11649743rs6983267 rs11649743 rs12418451 rs9623117 rs1859962 rs1571801 rs9623117rs11649743 rs5759167 rs9623117 rs7127900 rs5759167 rs1859962 rs5945619

1. A method of identifying a subject as having an increased risk ofdeveloping prostate cancer, comprising: a) determining, from a nucleicacid sample obtained from the subject, a genotype for the subject at aplurality of biallelic polymorphic loci, wherein each of said pluralityhas an associated allele and an unassociated allele, wherein thegenotype is selected from the group consisting of homozygous for theassociated allele, heterozygous, and homozygous for the unassociatedallele; and b) calculating a cumulative relative risk for the subjectbased on the genotype determined in step (a), wherein a cumulativerelative risk of greater than 1.0 identifies the subject as having anincreased risk of developing prostate cancer.
 2. The method of claim 1,wherein identification of the subject's increased risk of developingprostate cancer includes information about the subject's family history,prostate specific antigen (PSA) level, free to total PSA ratio, age,prostate volume, prior prostate biopsy history, number of previousbiopsy cores and/or family history.
 3. The method of claim 1, whereinthe subject has a family history of prostate cancer.
 4. The method ofclaim 1, wherein the subject has a prior negative prostate biopsy, 5.The method of claim 1, wherein identification of the subject as havingan increased risk of developing prostate cancer identifies the subjectas a candidate for prostate serum antigen (PSA) screening prior to age50.
 6. The method of claim 1, wherein identification of the subject ashaving an increased risk of developing prostate cancer identifies thesubject as a candidate for prostate biopsy.
 7. The method of claim 1,wherein identification of the subject as having an increased risk ofdeveloping prostate cancer identifies the subject, who is a subject witha prior negative prostate biopsy, as a candidate for subsequent prostatebiopsy.
 8. The method of claim 1, wherein identification of the subjectas having an increased risk of developing prostate cancer identifies thesubject as a candidate for chemopreventive therapy.
 9. The method ofclaim 1, wherein the plurality of biallelic polymorphic loci is amultiplicity, in any combination, of the single nucleotide polymorphismsof Table
 1. 10. The method of claim 1, wherein the plurality ofbiallelic polymorphis loci is the 33 single nucleotide polymorphisms ofTable 1.